Non-invasive devices and methods for detecting disease, such as diabetes, are described. In particular, the exemplary embodiments relate to methods and apparatuses suitable for determining in a mammal the presence, likelihood, progression and/or severity of diabetes mellitus.
Diabetes mellitus (“diabetes”) is a group of metabolic diseases in which a person has high blood sugar (hyperglycemia), either because the body does not produce enough insulin, or because the body's cells do not respond to the insulin that is produced. Diabetes is a disease derived from multiple causative factors and characterized by elevated levels of plasma glucose in the fasting state or after administration of glucose during an oral glucose tolerance test (OGTT). There are two primary forms of diabetes mellitus: (1) insulin dependent or Type 1 diabetes (a.k.a., Juvenile Diabetes, Brittle Diabetes, Insulin Dependent Diabetes Mellitus (IDDM)) and (2) non-insulin-dependent or Type II diabetes (a.k.a., NIDDM). Type 1 diabetes develops most often in young people, but can appear in adults that have the same auto anti body as the Type 1. Type 2 diabetes develops most often in middle aged and older adults, but can appear in young people. This high blood sugar condition produces symptoms of polyuria (frequent urination), polydipsia (increased thirst) or polyphagia (increased hunger). Diabetes is a large and growing problem throughout the world's developed and developing nations. As of now, it has been forecasted that approximately one in 10 U.S. adults have diabetes and according to a Centers for Disease Control and Prevention report, cases of diabetes are projected to double, even triple, by 2050 with as many as one in three having the disease, primarily type 2 diabetes.
Insulin is a hormone produced in the pancreas by β-cells. The function of insulin is to regulate the amount of glucose (sugar) in the blood, which enters cells through receptors that accept insulin and allow glucose to enter. Once inside a cell, glucose can be used as fuel. Excess glucose is stored in the liver and muscles in a form called glycogen. When blood glucose levels are low, the liver releases glycogen to form glucose. Without insulin, glucose has difficulty entering cells. In persons with diabetes mellitus, the pancreas either produces no insulin, too little insulin to control blood sugar, or defective insulin. Without insulin, these symptoms progress to dehydration, resulting in low blood volume, increased pulse rate, and dry, flushed, skin. In addition, ketones accumulate in the blood faster than the body is able to eliminate them through the urine or exhaled breath. Respiration becomes rapid and shallow and breath has a fruity odor. Other symptoms indicating a progression towards diabetic ketoacidotic coma (DKA) include vomiting, stomach pains, and a decreased level of consciousness. Persons with diabetes are at increased risk for debilitating complications such as renal failure, blindness, nerve damage and vascular disease. Although risk for or progression of complications can be reduced through tight glucose control combined with drug therapy and lifestyle changes, effective mitigation of complications begins with early detection. The disease leads to serious complications, including hyperglycemia, macroangiopathy, microangiopathy, neuropathy, nephropathy and retinopathy. As a result, diabetes adversely affects the quality of life. Similarly, uncontrolled Type 2 diabetes leads to excess glucose in the blood, resulting in hyperglycemia, or high blood sugar.
A person with Type 2 diabetes experiences fatigue, increased thirst, frequent urination, dry, itchy skin, blurred vision, slow healing cuts or sores, more infections than usual, numbness and tingling in feet. Without treatment, a person with Type 2 diabetes will become dehydrated and develop a dangerously low blood volume. If Type 2 diabetes remains uncontrolled for a long period of time, more serious symptoms may result, including severe hyperglycemia (blood sugar over 600 mg) lethargy, confusion, shock, and ultimately “hyperosmolar hyperglycemic non-ketotic coma.” Persistent or uncontrolled hyperglycemia is associated with increased and premature morbidity and mortality. As such, therapeutic control of glucose homeostasis, lipid metabolism, obesity, and hypertension are critically important in the clinical management and treatment of diabetes mellitus.
Pre-diabetes (i.e. where no overt clinical signs of diabetes are displayed) can be present for seven or more years before the detection of glycemic abnormalities and after disease onset and early stage diabetic complications are presented or diagnosed. More aggressive screening of individuals at risk for diabetes is needed. A major reason is that no simple and unambiguous laboratory test has existed that can be used to identify those subjects at risk for developing diabetes or pre-diabetes. There also is a need to identify subjects with a diabetic condition, including both pre-diabetic and diabetic subjects, so that they can obtain treatment early, and also to monitor the progression of the disease over time non-invasively. Early diagnosis, intensive treatment and consistent long-term follow-up evaluations for diabetic patients are essential for effective care, which can help preserve vision and significantly lower the risk of blindness. The diabetes Control and Complications Trial, (DCCT) in the USA demonstrated if a diabetic can be detected and brought under glucose control, complications can be reduced, e.g., (retinopathy) by eighty percent (80%). Once it becomes apparent that a patient may possibly develop diabetes, doctors are trained to ask the patient to return for more tests on a periodic basis to determine whether the patient's condition actually develops into the disease. Doctors have certain protocols about how long a patient should wait before being recalled for more testing. If a patient has few symptoms suggestive of diabetes, the patient may not be recalled from more than a year. If several suggestive symptoms are present, the doctor may wish to recall the patient after only a few months. Unfortunately, there is no diagnostic tool for accurately predicting how long a patient may have been experiencing diabetic symptoms, or for determining how great the patient's risk of actually developing the disease. If such a tool were available, it would enable a doctor to tailor his recall and therapy pattern to a patient's needs.
Modern diabetes screening and monitoring is a particularly “puncture-intensive” because diabetics have to draw blood to test their glucose levels. The only practical, reliable screening method currently available for monitoring blood glucose is by means of blood sampling. The primary screening and diagnostic tests currently in use—the Fasting Plasma Glucose (FPG) and the Oral Glucose Tolerance Test (OGTT)—are not considered to be optimum because they are inconvenient and unpleasant. Both require venous draws and are fasting tests so they can only be practically administered during morning appointments and are prone to non-compliance issues. For the OGTT, the measurement occurs two (2) hours after the patient ingests a 75 g oral glucose load. Numerous studies have evaluated the performance of each of these tests in diverse populations. It is believed that approximately one-half of those with diabetes are misclassified by a single FPG test. In addition, it is believed that the OGTT suffers from relatively poor reproducibility. In addition, the HbA1c test reflects longer term 90 day glycaemia and control or lack of control than FPG does, the results of the test can also be distorted due to recent changes in diet or hemolytic conditions. Such blood glucose measurement methodologies have limited value as indices of long-term glycemic status. In summary, blood glucose measurements (such as HbA1c and FPG) have limited value as reliable indices of long-term glycemic status.
Consequently, a rapid, accurate, reliable and convenient and non-invasive screening test is needed as a viable alternative to current tests. Ideally, an improved screening test would measure an analyte that is directly related to progression of the disease and the risk of complications, and the chemical marker would be invariant to within- or between-day changes in the patient as an integrated biomarker. In addition, the measurement should offer sufficient accuracy to detect diabetes in its early stages and possess adequate precision to eliminate the requirement for repeat, confirmatory testing. Once it becomes apparent that a patient may possibly have diabetes, doctors and optometrists will ask the patient to return for more tests on a periodic basis to determine whether the patient's condition actually develops into the disease or is confirmed to be diabetes. There are certain protocols about how long a patient should wait before being recalled for more testing. If a patient has few symptoms suggestive of diabetes, then the patient may not be recalled for more than a year. If several suggestive symptoms are present, then the patient may be recalled after only a few months. It would be useful if there was available a diagnostic tool and methods for non-invasively and accurately determining whether a patient is at risk of actually developing diabetes or actually has diabetes for immediate confirmation.
A major consequence of hyperglycemia is excessive glycosylation (non-enzymatic glycation) of proteins in a process known as the Maillard reaction. Excessive glycosylation eventually causes the formation of various protein-protein cross-links and non-crosslinked structures called Advanced Glycation End-products (AGEs). AGEs are believed to present an attractive candidate analyte for non-invasive measurements. AGEs have been implicated as causal factors in the complications of diabetes, including diabetic retinopathy (DR). Protein glycation is a multi-stage reaction that begins with formation of a sugar adduct to protein, known as a fructosamine or Amadori compound, which gradually matures to form AGEs. Some AGEs require oxidation chemistry for their formation and are known as glycoxidation products. Collagen is a protein that readily undergoes glycation and glycoxidation. Because of its long half-life, the level of AGEs in collagen is believed to act as a long-term integrator of overall glycemia that is insensitive to short- or intermediate-term fluctuations in glycemic control. As a result, AGEs accumulate naturally during healthy aging, but at significantly accelerated rates in persons with diabetes. Protein glycation and AGE formation are accompanied by increased free radical activity that contributes to the biomolecular damage in diabetes. Levels of AGEs are positively correlated with the severity of retinopathy, nephropathy and neuropathy and, as such are an indicator of systemic damage to protein in diabetes and a metric of a patient's risk for diabetic complications. In addition, due to the mild to severe hyperglycemia associated with pre-diabetes and type 2 diabetes, individuals who are in the early stages of this continuum will accumulate AGEs at higher than normal rates in their tissues. Thus, given sufficient assay sensitivity, an accurate AGE measurement in an individual offers the promise to detect early departure from normal glycemia. Currently, AGEs are assayed by invasive procedures requiring a biopsy specimen, and consequently are not used in diabetes screening or diagnosis.
Tissue such as the ocular lens can exhibit fluorescence when excited by a light source of a suitable wavelength. This fluorescence emission, arising from endogenous fluorophores, is an intrinsic property of the tissue and is called autofluorescence to be distinguished from fluorescent signals obtained by adding exogenous markers (like sodium fluorescein). The tissue fluorophores absorb certain wavelengths of light (excitation light), and release it again in light of longer wavelengths (emission). Several tissue fluorophores have been identified, such as collagen, elastin, lipofuscin, NADH, porphyrins and tryptophan. Each fluorophore has its characteristic excitation and emission wavelength, that enables localization and further quantification of a particular fluorophore. Autofluorescence can be induced in several tissues and can therefore be applied in investigation of several diseases. It is also used to distinguish malignant from benign tissue in several tissues, such as the skin and cervix. Furthermore, in ophthalmology, autofluorescence of the lens increases with ageing and diabetes. Autofluorescence of the lens appears to be caused by glycation and, subsequent oxidation of lens crystalline, which forms AGEs. The crystalline lens represents an exceptional bio target since the proteins in the lens are relatively static for life and do not turn over (i.e., undergo reverse glycation) allowing for the accumulation of AGEs.
Advances in fluorescence spectroscopy of the ocular lens has revealed a potential for a non-invasive device and method to sensitively measure changes in the lens of the eye associated with diabetes mellitus. The system relies on the detection of the spectrum of fluorescence emitted from a selected volume (about 1/10 mm3 to about 3 mm3 or more) of the lens of living human subjects using low power excitation illumination from monochromatic light sources. The sensitivity of this technique is based on the measurement of the fluorescence intensity in a selected region of the fluorescence spectrum and normalization of this fluorescence with respect to attenuation (scattering and absorption) of the incident excitation light. The amplitude of the unshifted Rayleigh line, measured as part of the fluorescence spectrum, is used as a measure of the attenuation of the excitation light in the lens. Using this methodology it is believed that the normalized lens fluorescence provides a more sensitive discrimination between diabetic and non-diabetic lenses than more conventional measurements of fluorescence intensity from the lens. Results from such clinical measurements could be used to describe a relationship between normalized lens fluorescence and hemoglobin A1c levels in diabetic patients.
Optical spectroscopy offers one potential avenue of early, non-invasive detection of diabetes by quantifying AGEs in the lens of the eye or other tissues. In spectroscopy, a machine fires a laser or other light on the skin or in the eye. Fluorescence spectroscopy (a.k.a. fluorometry or spectrofluorometry), is a type of electromagnetic spectroscopy that analyzes fluorescence from a sample by detecting the presence of certain molecules by measuring their reflected or emitted light. In fluorescence spectroscopy, the species is first excited, by absorbing a photon, from its ground electronic state to one of the various vibrational states in the excited electronic state. Collisions with other molecules cause the excited molecule to lose vibrational energy until it reaches the lowest vibrational state of the excited electronic state. The molecule then drops down to one of the various vibrational levels of the ground electronic state again, emitting a photon in the process. As different molecule species may drop down from different vibrational levels to the ground state, the emitted photons will have different energies, and thus frequencies. Those photons that are reflected from particles surfaces or refracted through them are called “scattered”. Scattered photons may encounter another grain or be scattered away from the surface so they may be detected and measured. Every molecule has a signature structure that reflects light at a specific wavelength; all glucose molecules share a unique signature that's entirely different from other blood components such as hemoglobin. If the returning wavelength differs from an established norm, the device alerts the patient or doctor to the presence of the molecule or cell in question. Therefore, by analyzing the different frequencies of light emitted in fluorescent spectroscopy, along with their relative intensities, the structure of the different vibrational levels can be determined.
Fluorescence-based systems rely on the propensity of certain cell components, known as fluorophores (e.g., tryptophan, flavins, collagen), to emit light when excited by specific wavelengths of light, with the peak intensity in a different, but corresponding frequency band. The actual amount of light emitted by fluorophores is exceedingly small (on the order of nanowatts) requiring an extremely sensitive photodetection system. The basic function of an optical spectroscopy device is to irradiate the specimen with a desired and specific band of wavelengths, and then to separate the much weaker emitted fluorescence from the excitation light. Only the emission light should reach the eye or detector so that the resulting fluorescent structures are superimposed with high contrast against a very dark (or black) background. The limits of detection are generally governed by the darkness of the background, and the excitation light is typically several hundred thousand to a million times brighter than the emitted fluorescence.
If AGEs are illuminated by light from 300-500 nm, then 400-700 nm fluorescence is emitted. Certain early metabolic changes may be detected by fluorescence spectroscopy as AGEs develop. Reflectance techniques attempt to characterize tissue by measuring the amount and wavelengths of light reflected back to a sensitive photodetector when the tissue (e.g., lens of the eye) is exposed to a light source. Fluorescence and reflected light measurements are analyzed using computer-based algorithms; however, these systems have not been studied extensively. Non-invasive ocular fluorescence measurements have been investigated on numerous occasions for diabetes screening and AGE quantitation.
For example, autofluorescence of the lens of the eye can be measured with a computer fluorophotometer (Fluorotron Master, Coherent Radiation Inc. (Palo Alto, Calif.)) fitted with a special lens (“anterior segment adapter”) for detailed scanning of lens. Autofluorescence of the lens, excited by a beam of continuous blue light can be scanned along the optical axis by moving the internal lens system of the fluorophotometer by a computer-controlled motor. The wavelengths of excitation and fluorescent light can be set by color filters with peak transmission at 490 nm and 530 nm respectively. The measured autofluorescence, expressed in equivalents of fluorescein concentration can be recorded as a function of distance in the eye.
It is always desirable to detect diseases early in their progress. In particular, it is desirable to screen and start treating glucose-intolerant individuals as early as possible since, even before the onset of diabetes, vascular lesions gradually develop with deterioration of glucose tolerance. Additionally, beta-cell function is seriously compromised by the time that overt alterations in glucose homeostasis, such as impaired glucose tolerance (IGT) and impaired fasting glucose (IFG), are manifest; thus, timely intervention is important to maintain residual insulin secretory capacity. Early detection enables early treatment which is generally believed to yield a higher success rate in treating various diseases. Recently, it is believed that analyzing eyes, and in particular the lenses of the eyes, can yield indications of various types of diseases. For example, measurements taken of light scattering within the eye has been shown to provide useful diagnostic information to detect and monitor the progress of diseases. Since this region is up to a few millimeters thick, measurements of this region, to be useful, need to be very accurate in the information for the position of the measurement. This is especially true because the human eye is in almost constant motion even when a patient is fixating on an illuminated target. This is particularly true because eye care professionals, such as optometrists, regularly examine, diagnose, treat and manage diseases, injuries, and disorders of the eyes and associated structures, as well as identify related systemic conditions affecting the eye. Optometrists, through their clinical education and experience, and broad geographic distribution, and the means to provide primary eye and vision care for the public. There often the first healthcare practitioners to examine patients with undiagnosed diabetes or ocular manifestations of diabetes.
The effectiveness of early intervention with lifestyle modification or medication in arresting disease progression has been demonstrated by the Diabetes Prevention Program (Diabetes Prevention Program Research Group. NEJM 346:393-403, 2002). However, the determination of IGT and IFG is itself an issue due to the relatively invasive nature of these assessments, particularly that of IGT by an oral glucose tolerance test (OGTT). In addition, an important additional diagnostic problem is monitoring of glucose homeostasis for confirming diabetes. Compliance with glucose monitoring is poor because of the pain and inconvenience of conventional blood collection using lancets. Furthermore, non-invasive monitoring techniques for diabetes, and to determine the efficacy of therapy, are desirable. Finally, assessment of progression of frank diabetes to complications is only feasible after complications are well established. Thus, it would be beneficial to have methods for assessing the development of diabetes from pre-diabetes, and for monitoring the course of the disease.
There is known at least one attempt to produce a commercial grade non-invasive diabetes detection/screening device that measures crystalline lens fluorescence, known as the Accu-Chek D-Tector. The Accu-Chek-D-Tector is essentially a confocal microscope in that it uses confocal optics to measure AGEs to check for early signs of uncontrollable blood sugar levels and type 2 diabetes because they build up more quickly in the eyes of individuals with high blood sugar levels than in the eyes of individuals with normal levels. The device employs so called biophotonic technology and detects diabetes by shining a blue light into the lens of the eye of a patient. The returned light is collected and analyzed. The light emitted from the eye of a person with diabetes is more intense than that of a person without diabetes. In particular, a laser beam passes through a light source aperture and then is focused by an objective lens into a small (ideally diffraction limited) focal volume within or on the surface of a patient's eye. Scattered and reflected laser light as well as any fluorescent light from the illuminated spot is then re-collected by the objective lens (collector). A beam splitter separates off some portion of the light into a detection apparatus, which in fluorescence confocal microscopy may have a filter that selectively passes the fluorescent wavelengths while blocking the original excitation wavelength. After optionally passing through a pinhole, the light intensity is detected by a photodetection device (e.g., a photomultiplier tube (PMT)), transforming the light signal into an electrical one that is recorded by a computer for further analysis. In particular, the Accu-Chek D-Tector shines a blue light into the lens of the eye, then collects and analyzes the returned light.
However, major drawbacks of the Accu-Chek-D-Tector are that it is relatively slow, imprecise and costly to manufacture. Although the device could purportedly take readings in 30 seconds (15 seconds for fluorescence, 15 seconds for backscatter) to obtain a ratio of fluorescence signal to backscattered signal from a specific location within the patient's lens, the device employed a sliding filter changer to select either green (fluorescence) or blue (backscattered) light striking a photodetector via a crank mechanism. Rotation of a step motor actuated the two position slider taking one or more seconds to move from one filter to the other. In addition, in use, the patient was required to self-align to the device via a fixation system that made it difficult and time-consuming.
Most non-invasive analyzers are not designed specifically for high-throughput screening purposes. They are difficult and expensive to integrate into a high-throughput screening environment. Even after the analyzer is integrated into the high-throughput screening environment, there often are many problems, including increased probability of system failures, loss of data, time delays, and loss of costly compounds and reagents. Thus, prior non-invasive diabetes detection devices generally have not recognized the need to provide analytic flexibility and high performance.
Typically, a non-invasive apparatus uses some form of spectroscopy to acquire the signal or spectrum from the body. Spectroscopic techniques include but are not limited to Raman and Rayleigh fluorescence, as well as techniques using light from ultraviolet through the infrared [ultraviolet (200 to 400 nm), visible (400 to 700 nm), near-infrared (700 to 2500 nm or 14,286 to 4000 cm-1), and infrared (2500 to 14,285 nm or 4000 to 700 cm-1)]. It is important to note, that these techniques are distinct from the traditional invasive and alternative invasive techniques listed above in that the sample analyzed is a portion of the human body in-situ, not a biological sample acquired from the human body.
A real need exists for a versatile, sensitive, high-throughput screening apparatus and methods that can handle multiple detections and wide ranges of patients while reliably maintaining a high level of sensitivity. In addition to early identification, it there is a need for diabetes detection apparatus, devices, methods and/or systems for detecting diabetes that requires no fasting and is a cumulative test that is not exposed to variations in glucose levels caused from a variety of reasons, including food, stress certain drugs, or short term changes in diet and exercise.